中国口腔颌面外科杂志 ›› 2020, Vol. 18 ›› Issue (4): 302-307.doi: 10.19438/j.cjoms.2020.04.003

• 论著 • 上一篇    下一篇

不同MEK1/2抑制剂对NF1敲低的施万细胞增殖活性的影响

刘嘉靓, 杜仲, 郑家伟, 游元和, 王延安   

  1. 上海交通大学医学院附属第九人民医院·口腔医学院 口腔颌面-头颈肿瘤科,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011
  • 收稿日期:2019-12-12 出版日期:2020-07-20 发布日期:2020-09-10
  • 通讯作者: 王延安,E-mail:wangyan_an@163.com
  • 作者简介:刘嘉靓(1993-),男,硕士,住院医师,E-mail:573078695@qq.com
  • 基金资助:
    国家自然科学基金(81371164,81870780)

Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells

LIU Jia-liang, DU Zhong, ZHENG Jia-wei, YOU Yuan-he, WANG Yan-an   

  1. Department of Oromaxillofacial Head and Neck Oncology,Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;
  • Received:2019-12-12 Online:2020-07-20 Published:2020-09-10

摘要: 目的: 构建慢病毒干扰载体介导的NF1 敲低RSC96(NF1kdRSC96)施万细胞株,对比不同MEK1/2抑制剂对该稳转细胞株增殖的影响。方法: 运用慢病毒干扰载体构建NF1kd的施万细胞模型,采用qRT-PCR及Western 免疫印迹验证慢病毒干扰载体组(NF1kd组)和空白载体组的NF1表达水平;应用5种MEK1/2抑制剂,包括司美替尼(Selumetinib)、贝美替尼(Binimetinib)、考比替尼(Cobimetinib)、曲美替尼(Trametinib)、PD0325901处理细胞,CCK-8法检测不同MEK1/2抑制剂在时间梯度和浓度梯度下的抑制作用。使用Graphpad Prism 5计算NF1kd组和空白载体组中5种抑制剂的半抑制浓度(IC50)值,采用SPSS 22.0软件包对数据进行统计学分析。结果: 5种MEK1/2抑制剂均对NF1kd施万细胞的增殖有抑制作用(P<0.05)。72 h时,PD0325901、Cobimetinib和Trametinib 3种抑制剂的抑制作用显著强于Selumetinib和Binimetinib(P<0.05)。IC50实验中,Cobimetinib抑制NF1kd组及空白载体组的药物浓度分别为(2.35±0.98)μmol/L和(14.22±1.67) mol/L,组间差异显著(P<0.05);Binimetinib抑制NF1kd组及空白载体组的药物浓度分别为(18.96±2.37) mol/L和(114.1±5.98) mol/L,组间差异显著(P<0.05),而Selumetinib、Trametinib和PD0325901抑制NF1kd组及对照组的IC50值无显著差异(P>0.05)。结论: 成功构建了NF1敲低的施万细胞稳转株。5种MEK1/2抑制剂均能抑制NF1kdRSC96稳转细胞株的增殖活性,PD0325901、Trametinib和Cobimetinib具有更持久的抑制作用。相同药物浓度下,Cobimetinib和Binimetinib能更特异地抑制NF1kd施万细胞的增殖活性。Cobimetinib同时具备药效持久性和抑制特异性,可能具有一定的临床应用前景。

关键词: NF1, 慢病毒, 施万细胞, MEK1/2抑制剂, I型神经纤维瘤

Abstract: PURPOSE: To establish a stable RSC96 cell line with knockdown of neurofibromin 1(NF1) gene by lentiviral vectors, and assess the influence of different MEK1/2 inhibitors on proliferation of NF1 knockdown RSC96 Schwann cell. METHODS: Lentivirus was used to construct a Schwann cell model with decreased expression of NF1 gene. qRT-PCR and Western blot (WB) were used to verify the expression level of NF1 gene. Five MEK1/2 inhibitors were included in this experiment: Selumetinib, Binimetinib, Cobimetinib, Trametinib and PD0325901. CCK-8 assay was used to detect the inhibition of different MEK1/2 inhibitors under time gradient and concentration gradient. IC50 values in the blank control group and NF1kd group of 5 inhibitors were calculated by Graphpad Prism software package. SPSS 22.0 software package was used for statistical analysis. RESULTS: All MEK1/2 inhibitors could inhibit the proliferation of NF1kd Schwann cells (P<0.05). At 72 h, PD0325901, Cobimetinib and Trametinib had stronger inhibitory effects than Selumetinib and Binimetinib (P<0.05). In IC50 experiment, the drug concentrations of Cobimetinib in NF1kd group and blank control group were (2.35±0.98) mol/L and (14.22±1.67) mol/L, respectively(P<0.05); the drug concentrations of Binimetinib in NF1kd group and blank control group were (18.96±2.37) mol/L and (114.1±5.98) mol/L, respectively; and the differences between the groups were significant (P<0.05). There was no significant difference in IC50 values within Selumetinib, Trametinib and PD0325901 in NF1kd group and control group (P>0.05). CONCLUSIONS: Stable Schwann cell line with knockdown of NF1 gene was successfully constructed. All five MEK inhibitors can inhibit the proliferation of NF1kd RSC96 cell line; PD0325901, Trametinib and Cobimetinib processed longer lasting inhibiting ability. Cobimetinib and Binimetinib can both kill NF1kd Schwann cells more specifically than the blank control group at the same drug concentration. Cobimetinib has both long-lasting efficacy and killing specificity, which may have more clinical prospects.

Key words: NF1, Lentivirus, Schwann cells, MEK1/2 inhibitors, Neurofibromatosis type 1

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